Contributed equally
Resistance to conventional chemotherapeutic agents, including irinotecan (CPT-11), 5-fluorouracil and capecitabine is a major cause for therapeutic failure in patients with colorectal cancer (CRC). Increasing evidence has demonstrated that cancer cells exhibiting stem cell-like characteristics are associated with the development of resistance to chemotherapeutic agents. As a plant polyphenol, curcumin has been demonstrated to have the ability to ameliorate resistance of CRC to chemotherapeutic agents, but the associations among curcumin, cancer stem cells (CSCs) and chemoresistance of CRC remain unclear. The present study established a CPT-11-resistant colon cancer cell line, LoVo/CPT-11 cells, and detected the expression levels of CSC identification markers [cluster of differentiation (CD)44, CD133, epithelial cell adhesion molecule (EpCAM) and CD24] in parental cells and CPT-11-resistant cells. It was revealed that the expression levels of the colon CSC markers in LoVo/CPT-11 cells were significantly higher compared those in parental cells at the mRNA and protein level. The effect of curcumin on the chemoresistance to CPT-11 and the expression levels of CSC identification markers in LoVo/CPT-11 cells separately treated with curcumin and CPT-11 were further investigated. The results revealed that curcumin significantly attenuated chemoresistance to CPT-11, and treatment with curcumin resulted in a significant reduction of the expression levels of CSC identification markers. Furthermore, a tumor sphere formation assay was used to enrich colon CSCs from LoVo/CPT-11 cells, and demonstrated that curcumin efficiently diminished the traits of colon CSCs, as evidenced by the inability to form tumor spheres, the reduction in the expression of CSC identification markers, and apoptosis-induced effects on sphere-forming cells treated with curcumin alone or in combination with CPT-11. Altogether, the present data demonstrated that curcumin attenuated resistance to chemotherapeutic drugs through induction of apoptosis of CSCs among colon cancer cells. These findings may provide novel evidence for the therapeutic application of curcumin in CRC intervention.
In the last decade, the incidence and mortality rates of colorectal cancer (CRC) have decreased to a certain extent owing to improved screening, diagnostic and therapeutic technical expertise for cancer treatment. Despite these positive developments, CRC remains one of the most common cancer types and the primary cause for cancer-associated mortality worldwide (
Curcumin, a dietary polyphenolic compound extracted from
Based on the aforementioned observations, CPT-11-resistant cells and sphere-forming cells were developed in the present study to explore the effects of curcumin on chemoresistance. Following the induction of resistance to CPT-11 in LoVo cells, the changes in the expression levels of drug-resistant-associated proteins and CSC identification markers were detected. Next, the extent of chemoresistance and the expression levels of CSC markers in CPT-11-resistant cells were evaluated following treatment with curcumin. Furthermore, the effect of curcumin on tumor sphere formation in colon CSCs, as well as the expression levels of CSC identification markers in sphere-forming cells were investigated. Finally, whether curcumin targets CSCs via induction of apoptosis was explored.
Human colon cancer LoVo cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% penicillin/streptomycin (Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China) in a humidified 37°C incubator with 5% CO2. CPT-11 (Jiangsu Hengrui Medicine Co., Ltd., Lianyungang, China)-resistant cells, referred to as LoVo/CPT-11 cells, were established by repetitive treatment of the LoVo cells with increasing concentrations of CPT-11 over a 10–12-month period, based on the methods described in our previous studies (
Primary rabbit polyclonal antibodies against cluster of differentiation (CD)133 (cat. no. 18470-1-AP), CD44 (cat. no. 15675-1-AP), epithelial cell adhesion molecule (EpCAM; cat. no. 21050-1-AP), CD24 (cat. no. 18330-1-AP) and β-actin (cat. no. 20536-1-AP) were purchased from ProteinTech Group, Inc. (Chicago, IL, USA). Rabbit poly-clonal antibodies against ATP binding cassette subfamily B member 1 (ABCB1; cat. no. ab155421), cleaved caspase-3 (cat. no. ab13847), cleaved caspase-9 (cat. no. ab2324), cleaved caspase-8 (cat. no. ab25901), BCL2 associated X apoptosis regulator (Bax; cat. no. ab53154), apoptosis regulator Bcl-2 (Bcl-2; cat. no. ab196495), and the horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (cat. no. ab7090), were purchased from Abcam (Cambridge, UK). Curcumin was purchased from Merck KGaA (Sigma-Aldrich, Darmstadt, Germany).
A Cell Counting kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was used to assess the inhibition of cell growth in response to CPT-11 (0, 5, 10, 20, 40, 80, 120, 160 and 200
LoVo/CPT-11 cells were cultured in serum-free Dulbecco's modified Eagle's medium/F12 (Gibco; Thermo Fisher Scientific, Inc.) with 20 ng/ml epidermal growth factor, 10 ng/ml basic fibroblast growth factor (all from PeproTech, Inc., Rocky Hill, NJ, USA), 2% B27 supplement (Thermo Fisher Scientific, Inc.), and 5
The percentages of CD133-positive cells in LoVo, LoVo/CPT-11 and sphere-forming cells were measured using a flow cytometry assay. Briefly, cells were separately centrifuged (4°C, 5 min, 100 × g) and washed with ice-cold PBS. Subsequently, 1×106 cells were incubated with 5
To analyze the effect of curcumin on the chemoresistance of LoVo/CPT-11 cells, different concentrations of curcumin (0, 2.5 and 5
Apoptosis distribution was detected using a fluorescein isothiocyanate Annexin V Apoptosis Detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol. In brief, sphere-forming cells were treated with curcumin and CPT-11 individually and in combination for 24 h, then cells were collected and washed with cold PBS twice. Subsequently, the cells were resuspended in 100
Total cellular mRNA from LoVo, LoVo/CPT-11 and sphere-forming cells were extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Subsequently, 500 ng of purified mRNA was reverse-transcribed into cDNA following the manufacturer's protocol (Beijing Transgen Biotech Co., Ltd., Beijing, China). The qPCR amplification was performed in triplicate using a Light Cycler® 480 II (Roche Applied Science, Penzberg, Germany). The reaction system (20
Cells were collected using a plastic scraper, washed with cold PBS and then solubilized in ice-cold protein extract solution RIPA containing protease inhibitors (Beyotime Institute of Biotechnology, Shanghai, China). The supernatant was used for western blot analysis following clarification. Protein concentration was determined using a BCA kit (Beyotime Institute of Biotechnology) and standardized between the samples. Equal amounts (30
All data are expressed as the mean ± standard deviation of three independent experiments. Statistical comparisons were determined using unpaired t-tests or one-way analysis of variance with Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. All statistical calculations were performed using SPSS software (version 20.0; IBM Corp., Armonk, NY, USA), Graphs were prepared using GraphPad Prism software (version 5.0; GraphPad Software, Inc., La Jolla, CA, USA).
Via long-term culturing of original CPT-11-sensitive LoVo cells by gradual adaptation to increasing CPT-11 concentrations, CPT-11-resistant LoVo cells were successfully established. The resistance to CPT-11 in LoVo/CPT-11 cells was examined by treating these cells with different concentrations of CPT-11. The growth inhibitory rates were compared with those of their parental cells using a CCK-8 assay. The results demonstrated that the growth inhibitory rates of LoVo/CPT-11 cells treated with CPT-11 were significantly decreased when compared with the parental cells from concentration ≥5
Curcumin inhibited the growth of LoVo/CPT-11 cells in a concentration-dependent manner. At 2.5 and 5
To further explore the possibility that curcumin attenuated chemoresistance via reducing CSC-like characteristics, LoVo/CPT-11 cells were treated with different concentrations of curcumin and CPT-11 individually for 24 h. The results revealed that, with the exception of CD133 mRNA expression, the protein expression of CD133, and the mRNA and protein expression levels of CD44, EpCAM and CD24 were inhibited by curcumin in a concentration-dependent manner (
LoVo/CPT-11 cells were plated in SFM containing several appropriate growth factors in order to form stable cell spheroids. In order to verify the CSC-like characteristics of sphere-forming cells, the percentages of CD133-positive cells were detected. As demonstrated in
To detect the effect of curcumin and CPT-11 on colon CSCs, sphere-forming cells were treated with curcumin and CPT-11 separately or in combination for 3 days. As shown in
To further to understand the effect of curcumin on colon CSCs, the effects of curcumin on apoptosis in sphere-forming cells were investigated. The results of flow cytometry analysis indicated that cell apoptosis was induced in the presence of curcumin in sphere-forming cells, and the apoptosis-inducing effect of combined curcumin and CPT-11 was significantly increased compared with that of curcumin alone (
CRC is the fourth leading cause of cancer-associated mortalities worldwide, and is associated with an unsatisfactory prognosis and <10% rate of 5-year survival rate (
In the present study, a CPT-11-resistant cell subline was derived from the human colon cancer LoVo cell line, which was confirmed to exhibit increased cell viability compared with of the parental cell line in the presence of different concentrations of CPT-11. A previous study demonstrated that ABC transporters contribute to the chemoresistance by pumping anticancer agents out of the cells (
CPT-11 combined with 5-fluorouracil is one of the standard chemotherapeutic options for metastatic CRC. Nevertheless, the effectiveness of CPT-11 and other agents is restricted as they ineffectively target CSCs, which contributes to the development of chemoresistance (
Several methods have been reported to isolate CSCs, including the side population (SP) cell sorting method, magnetic activated cells sorting (MACS), fluorescence activated cells sorting (FACS) and SFM culture (
The association between the inhibitory effect of curcumin and apoptosis was further explored. The results of the flow cytometry assay suggested that curcumin significantly induced apoptosis of CSCs, while CPT-11 had no significant effect on induction of apoptosis. Furthermore, curcumin in combination with CPT-11 demonstrated a stronger effect compared with curcumin alone. Curcumin may not only be an 'effector' itself, but also serve as a 'catalyzer' for CPT-11 in inducing apoptosis, resulting in a synergistic effect between curcumin and CPT-11. This may be investigated in future studies. In order to achieve higher credibility, western blot assays were used to verify the effect of curcumin on the induction of apoptosis. As a family of cysteine aspartic acid-specific proteases, caspases are essential components of apoptotic pathways, which include the mitochondrial and death receptor pathways (
Several other cellular signaling pathways are known to regulate cell apoptosis and proliferation, and we hypothesize that the phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase (AKT), mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) signaling pathways are associated with the effects of curcumin observed in the present study. The PI3K/AKT signaling pathway is dysregulated in numerous types of human cancer, and regulates the apoptotic response via interaction with the key mediators of the apoptotic process (
Taken together, these findings suggest that CSCs serve an important role in the development of chemoresistance in CRC cells. The results of the present study indicated that the natural compound curcumin effectively attenuated chemoresistance of CRC cells via targeting and inducing apoptosis in CSCs, hence eliminating CSCs. The current study provides evidence for the clinical therapeutic potential of curcumin as a supplement to conventional chemotherapy in patients with CRC experiencing resistance to conventional anticancer drugs.
colorectal cancer
cancer stem cell
cluster of differentiation 133
cluster of differentiation 44
cluster of differentiation 24
epithelial cell adhesion molecule
irinotecan
curcumin
The authors would like to thank the public experimental platform (Research Center of Clinical Medicine of Nanfang Hospital Affiliated to Southern Medical University, Guangzhou, Guangdong, China) for providing experimental facilities.
The present study was supported by the China Postdoctoral Science Foundation (grant no. 2015M582358, received by GW) and the Project of Traditional Chinese Medicine Bureau of Guangdong Province (grant no. 20141307, received by XC).
The analyzed datasets generated during the study are available from the corresponding author on reasonable request.
XC and PS conceived and designed the experiments. PS and YY performed the experiments and acquired data. YJ and GW provided the methodology, analyzed and interpreted the data. PS and YY drafted the manuscript. YJ directed the writing of the manuscript and revised the manuscript. YJ and PS critically revised the manuscript. All authors have given final approval of the version to be published.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
Growth inhibitory rates, and the expression of ABCB1 and CSC identification markers in LoVo and LoVo/CPT-11 cells. (A) The growth inhibitory rates of LoVo and LoVo/CTP-11 cells treated with various concentrations of CPT-11 for 24 h, as determined using a cell counting kit-8 assay. (B) Representative western blotting image, (C) quantification of protein expression and (D) mRNA expression of ABCB1 in LoVo and LoVo/CPT-11 cells. (E) Representative western blotting image, (F) quantification of protein expression and (G) mRNA expression of CSC identification markers in LoVo and LoVo/CPT-11 cells. Relative protein expression was measured using ImageJ. β-actin was used as a loading control. Each bar represents the mean ± standard deviation of three independent experiments. *P<0.05 and ***P<0.001 vs. LoVo cells. 1, LoVo cells; 2, LoVo/CPT-11 cells. ABCB1, ATP-binding cassette Subfamily B Member 1; CSC, cancer stem cell; CPT-11, irinotecan.
Growth inhibitory rates and the changes in CSC markers in LoVo/CPT-11 cells following various treatments. The growth inhibitory rates of LoVo/CTP-11 cells treated with (A) curcumin alone or (B) a combination of CPT-11 and curcumin for 24 h was determined using a cell counting kit-8 assay. (C) Representative western blotting image, (D) quantification of protein expression and (E) mRNA expression of CSC identification markers in LoVo/CPT-11 cells treated with curcumin. (F) Representative western blotting image, (G) quantification of protein expression and (H) mRNA expression of CSC identification markers in LoVo/CPT-11 cells treated with CPT-11. β-actin was used as a loading control. Each bar represents the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. untreated control cells. CSC, cancer stem cell; CPT-11, irinotecan.
Percentages of CD133-positive cells in LoVo, LoVo/CPT-11 and sphere-forming cells. Each bar represents the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001. PE-A, PE-conjugated anti-CD133 antibody. CPT-11, irinotecan; PE, P-phycoerythrin; CD133, cluster of differentiation 133.
Changes in morphology and the number of tumor spheres in sphere-forming cells. LoVo/CPT-11 cells were plated as single cells in ultra-low attachment 6-well plates at 2.0×104 cells/well. Then, the cells were treated separately on the third day with various concentrations of CPT-11 and curcumin. The tumor sphere changes were observed under a light microscope 3 days later. (Original magnification, ×100). The morphology changes in sphere-forming cells (A) CPT-11, (B) curcumin and (C) combined treatments. The normalized sphere-formation efficiency following (D) CPT-11, (E) curcumin and (F) combined treatments. Each bar represents the mean ± standard deviation of the three independent experiments. ***P<0.001 vs. control cells. CPT-11, irinotecan.
Changes in CSC identification markers in sphere-forming cells following various treatments. The protein expression (A) and the relative protein expression (B) and the mRNA expression (C) of CSC markers in sphere-forming cells in different groups. β-actin was used as a loading control. Each bar represents the mean ± standard deviation of the three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control cells. CSC, cancer stem cell.
Rates of apoptosis and the expression of apoptosis-associated proteins in sphere-forming cells. (A) The flow cytometry profiles and (B) quantitative analysis of the apoptosis rate of spheroid-forming cells following treatment with curcumin and CPT-11 separately and in combination. (C) Representative blot of apoptosis-associated protein expression and (D) quantification of protein expression in spheroid-forming cells following treatment with curcumin and CPT-11 separately and in combination. β-actin was used as a loading control. Each bar represents the mean ± standard deviation of the three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control cells. CPT-11, irinotecan.